DNA-protein cross-linking by 1,2,3,4-diepoxybutane

Abstract

1,2,3,4-diepoxybutane (DEB) is a strongly genotoxic diepoxide hypothesized to be the ultimate carcinogenic metabolite of the common industrial chemical and environmental carcinogen 1,3-butadiene. DEB is a bis-electrophile capable of cross-linking cellular biomolecules to form DNA-DNA and DNA-protein cross-links (DPCs), which are thought to play a central role in its biological activity. Previous studies with recombinant proteins have shown that the biological outcomes of DEB-induced DPCs are strongly influenced by protein identities. The present work combines affinity capture methodology with mass spectrometry-based proteomics and immunological detection to identify the proteins that form DPCs in nuclear extracts from human cervical carcinoma (HeLa) cells. We identified 39 human proteins that form covalent DPCs in the presence of DEB. DNA-protein cross-linking efficiency following treatment with 25 mM DEB was 2-12%, depending on protein identity. High-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI+-MS/MS) analysis of the total proteolytic digests of cross-linked proteins revealed the presence of 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol conjugates, suggesting that DEB forms DPCs between cysteine thiols within proteins and the N-7 guanine positions within DNA.

Figure 1

Concentration-dependent formation of DPCs following DEB exposure of nuclear protein extracts prepared from HeLa cells in the presence of DNA. (A) Nuclear protein extracts from HeLa cells (500 μg) and 5′-biotinylated double-stranded oligodeoxynucleotides (3.12 nmol) were incubated in the presence of 0 (lane 2), 5 (lane 3), 10 (lane 4), 25 (lane 5), 50 (lane 6), or 100 mM DEB (lane 7). The resulting DPCs were subjected to biotin capture enrichment, hydrolyzed to release protein-guanine conjugates, and resolved by 12% SDS-PAGE. Gels were stained with SimplyBlue SafeStain to visualize the cross-linked proteins. (B) Densitometric analysis of protein bands in the 25 – 250 kDa molecular weight range to estimate the extent of total protein cross-linking to DNA in the presence of DEB. Band intensity was compared to staining of a known amount of nuclear protein extract analyzed as a control to estimate the cross-linking efficiency.

Figure 2

Examples of HPLC-ESI⁺-MS/MS spectra of tryptic peptides used for protein identification: Fen-1 (A), GAPDH (B), and PARP (C) present in affinity-captured DPCs. ^‡Cysteine carboxamidomethylation (+57).

Figure 3

Cellular functions of human proteins that form DPCs in the presence of DEB, as identified by affinity capture in combination with mass spectrometry-based proteomics.

Figure 4

Western blot analysis of DEB-induded DPCs in nuclear protein extracts from HeLa cells. (A) Nuclear extract proteins were incubated with 0 (lane 1), 5 (lane 2), 10 (lane 3), or 25 mM DEB (lane 4) in the presence of 5′-biotinylated double-stranded oligodeoxynucleotides (5′-GGA GCT GGT GGC GTA GGC-3′ (+) strand). Following biotin capture enrichment and hydrolysis to release protein-guanine conjugates, the cross-linked proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Western blots were performed using primary antibodies against actin, AGT, GAPDH, PARP, and Ref-1. (B) Densitometric analysis of western blots to estimate the extent of protein cross-linking to DNA in the presence of DEB.

Figure 5

HPLC-ESI⁺-MS/MS analysis of 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol (Cys-N7G-BD) conjugates in total proteolytic digests of DEB-induced DPCs. Nuclear protein extracts from HeLa cells were exposed to DEB in the presence of biotinylated DNA duplexes. Following biotin capture enrichment, DPCs were subjected to thermal hydrolysis and enzymatic digestion of proteins to amino acids to release amino acid-nucleobase conjugates: (A) Synthetic Cys-N7G-BD; (B) enzymatic digests of HeLa nuclear protein extracts following incubation with DNA in the absence of DEB (negative control); (C) enzymatic digests of HeLa nuclear protein extracts following incubation with 50 mM DEB in the presence of DNA.

Scheme 1

Metabolic activation of BD to DEB and DEB-mediated formation of DNA-protein cross-links.

Scheme 2

Experimental scheme for biotin capture enrichment of DPCs from nuclear protein extracts incubated with DEB in the presence of double-stranded DNA.